• Home
  • Search Results
  • Generation of food-grade recombinant Lactobacillus casei delivering Myxococcus xanthus prolyl endopeptidase.

Generation of food-grade recombinant Lactobacillus casei delivering Myxococcus xanthus prolyl endopeptidase.

Applied microbiology and biotechnology (2014-04-23)
Patricia Alvarez-Sieiro, Maria Cruz Martin, Begoña Redruello, Beatriz Del Rio, Victor Ladero, Brad A Palanski, Chaitan Khosla, Maria Fernandez, Miguel A Alvarez
ABSTRACT

Prolyl endopeptidases (PEP) (EC 3.4.21.26), a family of serine proteases with the ability to hydrolyze the peptide bond on the carboxyl side of an internal proline residue, are able to degrade immunotoxic peptides responsible for celiac disease (CD), such as a 33-residue gluten peptide (33-mer). Oral administration of PEP has been suggested as a potential therapeutic approach for CD, although delivery of the enzyme to the small intestine requires intrinsic gastric stability or advanced formulation technologies. We have engineered two food-grade Lactobacillus casei strains to deliver PEP in an in vitro model of small intestine environment. One strain secretes PEP into the extracellular medium, whereas the other retains PEP in the intracellular environment. The strain that secretes PEP into the extracellular medium is the most effective to degrade the 33-mer and is resistant to simulated gastrointestinal stress. Our results suggest that in the future, after more studies and clinical trials, an engineered food-grade Lactobacillus strain may be useful as a vector for in situ production of PEP in the upper small intestine of CD patients.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Chloramphenicol, ≥98% (HPLC)
Sigma-Aldrich
Ampicillin, anhydrous, 96.0-102.0% (anhydrous basis)
Sigma-Aldrich
Erythromycin, BioReagent, suitable for cell culture
Sigma-Aldrich
Chloramphenicol, BioReagent, suitable for plant cell culture
Sigma-Aldrich
Lactose, tested according to Ph. Eur.
Supelco
Ampicillin, analytical standard
Sigma-Aldrich
Ampicillin, meets USP testing specifications
Supelco
Erythromycin, Pharmaceutical Secondary Standard; Certified Reference Material
Supelco
Chloramphenicol, VETRANAL®, analytical standard
Sigma-Aldrich
Erythromycin, potency: ≥850 μg per mg
Sigma-Aldrich
Maltose solution, BioReagent, for molecular biology, ~20% in H2O
Sigma-Aldrich
Erythromycin, meets USP testing specifications
USP
Erythromycin, United States Pharmacopeia (USP) Reference Standard
Sigma-Aldrich
Chloramphenicol, meets USP testing specifications
Sigma-Aldrich
Chloramphenicol, γ-irradiated
Sigma-Aldrich
Erythromycin standard solution, 1 mg/mL in H2O
USP
Ampicillin, United States Pharmacopeia (USP) Reference Standard
Erythromycin, for microbiological assay, European Pharmacopoeia (EP) Reference Standard
Ampicillin, anhydrous, European Pharmacopoeia (EP) Reference Standard
Sigma-Aldrich
L-Proline p-nitroanilide trifluoroacetate salt, prolyl aminopeptidase substrate
Chloramphenicol, European Pharmacopoeia (EP) Reference Standard
Sigma-Aldrich
Erythromycin, tested according to Ph. Eur.